Devices for Flow Step Focusing (FSF)

ABSTRACT

Flow step focusing isolates and concentrates a molecule of interest by flowing a liquid comprising a molecule of interest through a main channel having an inlet and an outlet with application of a first pressure at the inlet; applying a voltage along the channel during the flowing, wherein the voltage is configured to have a polarity such that it drives the molecule of interest in a direction opposite the flow of the liquid; controlling the first pressure and/or the voltage in a manner so as to trap and concentrate the molecule of interest in a region of the main channel; and removing the concentrated molecule of interest from the channel by recovering a portion of the liquid from a side channel diverging from the main channel, wherein the side channel is maintained at a pressure lower than the first pressure. Also disclosed is an apparatus for such.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit as a divisional application of U.S.patent application Ser. No. 13/625,585 filed on Sep. 24, 2012 which inturn claims the benefit of U.S. Provisional Application 61/540,081 filedon Sep. 28, 2011.

BACKGROUND

Many facets of modern medicine and science depend on the ability toreliably measure the presence and quantity of compounds in solution. Itremains difficult to differentiate a signal of interest arising from asingle compound in a complex mixture of similar compounds. The problemis particularly severe in biological systems, as a molecule of interestin will often be part of a family of closely related compounds thatappear similar (if not identical) to the detector.

Several techniques exist that can separate compounds out of a complexmixture so that they can be measured. Chromatography is a well-knownexample, wherein a small sample of the mixture is injected into a columnof packed separation media-, and flushed through with an appropriatesolvent. The compounds dissolved in the sample migrate down the columnat different speeds and leave the far end at different times. However,the fractions collected at the end of the column inevitably have a muchgreater volume than the original sample. Dilution is the price paid forbeing able to tease the compound of interest away from its interferingrelatives. While this tradeoff may be acceptable in many circumstances,when our samples are small, or the compound of interest is alreadydilute, the dilution of chromatographic separation is a serious problem.

Techniques to both isolate and concentrate solutes for analysis arerarely done because they are often cumbersome, multistep processes. Theytypically involve a separation step (e.g. chromatography with fractioncollection) followed by a laborious concentration step, often involvingevaporating or subliming away the solvent. It is seldom done because itcostly, time consuming, requires trained technicians, and is difficultto do quantitatively.

Focusing techniques can perform both the isolation and concentration inone step. They most commonly depend upon the molecules in question beingsubjected to two opposing forces. For example, in gradientelectrofocusing, the sample solution flows through a non-uniformelectric field. The drag of the flowing fluid provides the first drivingforce on the molecules, and does not change over the length of thechannel. The electric field drives the molecules against the directionof flow, and becomes stronger as they move down the channel. There is apoint in the channel where the two driving forces become equal. This iswhere the molecules will become focused.

Focusing within a flowing channel has be demonstrated using a variety ofmethods, including electric field gradient focusing (EFGF), temperaturegradient focusing (TGF) and isoelectric focusing (IEF). These lack aside channel to allow removal of the concentrated band. A variant ofIEF, known as free-flow IEF, allows for a continuous separation (see Wenet al.). In the “free flow” format, focusing is performed perpendicularto the direction of flow. The degree of concentration is necessarilylimited because solutes can only spend a finite time in the channelbefore being flushed out. There is no “trap-and-hold” capability.

BRIEF SUMMARY

In one embodiment, a method of isolating and concentrating a molecule ofinterest includes flowing a liquid comprising a molecule of interestthrough a main channel having an inlet and an outlet with application ofa first pressure at the inlet; applying a voltage along the channelduring the flowing, wherein the voltage is configured to have a polaritysuch that it drives the molecule of interest in a direction opposite theflow of the liquid; controlling the first pressure and/or the voltage ina manner so as to trap and concentrate the molecule of interest in aregion of the main channel; and removing the concentrated molecule ofinterest from the channel by recovering a portion of the liquid from aside channel diverging from the main channel, wherein the side channelis maintained at a pressure lower than the first pressure.

In another embodiment, an apparatus configured to isolate andconcentrate a molecule of interest includes a main channel having aninlet and an outlet; a side channel intersecting the main channel andhaving a side channel outlet; a first pressure controller operablyconnected to the inlet and configured for pumping fluid into the inlet;a second pressure controller operably connected to either the mainchannel outlet or the side channel outlet and configured to apply apressure or a vacuum thereto; and electrodes at each of the inlet andthe outlet operably connected to a voltage controller configured togenerate a voltage between the electrodes so as to generate an electricfield along the channel.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an exemplary channel configured forflow step focusing.

FIG. 2 is a schematic illustration of an exemplary apparatus for flowstep focusing.

FIG. 3 is a series of images taken sequentially (top to bottom) of aband moving from right to left across an intersection with a sidechannel.

DETAILED DESCRIPTION

Definitions

Before describing the present invention in detail, it is to beunderstood that the terminology used in the specification is for thepurpose of describing particular embodiments, and is not necessarilyintended to be limiting. Although many methods, structures and materialssimilar, modified, or equivalent to those described herein can be usedin the practice of the present invention without undue experimentation,the preferred methods, structures and materials are described herein. Indescribing and claiming the present invention, the following terminologywill be used in accordance with the definitions set out below.

As used in this specification and the appended claims, the singularforms “a”, “an,” and “the” do not preclude plural referents, unless thecontent clearly dictates otherwise.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items.

As used herein, the term “about” when used in conjunction with a statednumerical value or range denotes somewhat more or somewhat less than thestated value or range, to within a range of ±10% of that stated.

As used herein, the term “molecule of interest” can includes particles,nanoparticles, and cells, as well as molecules. To effectively functionas molecules of interest in flow step focusing, they must have a charge,either natively or as provided by complexing them with buffer additivesin a liquid.

Description

Flow step focusing is a technique by which a molecule of interest can beboth separated and concentrated in a single step. A liquid containing amolecule of interest is driven through a channel (e.g., using a pump)while an electric voltage is applied along the channel. The voltageshould have a polarity such that it drives the molecule of interest in adirection opposite the flow of the liquid. With flow velocity higher onthe upstream end of the channel than the downstream end, molecules withan electrophoretic velocity in between the upstream and downstream flowvelocities will move downstream on the upstream end, and upstream on thedownstream end. Thus, molecules of interest become trapped in a narrowregion (a “band”) within the channel. Over time, the concentrationwithin the region increases as more molecules are added. The position ofthe band can be adjusted by changing either the voltage or appliedpressure (a pressure change operating to change the flow rate). Aportion or all of the band can periodically pulled off by moving itacross an intersection with a side channel, where fluid is removed fromthe channel through a side channel outlet.

Operationally, the pressure should be greatest at the inlet of the mainchannel and lowest at the outlet, with the side channel at anintermediate pressure, regardless of relation to ambient pressure. Inthe examples, the outlet was at ambient pressure and the other portspressurized. Alternatively, one could also apply a positive pressure tothe inlet and a negative to the outlet, leaving the side at ambient. Itis also possible to apply negative pressure (partial vacuum) to the mainchannel outlet and side channel outlet, leaving the inlet at ambientpressure. Accordingly, the pressure controllers as decribed hereininclude those supplying positive and/or negative pressure. As known to aperson of skill in the art, resistance can be controlled through thedesign of main and side channel dimensions as well as through pumps,regulators, and the like.

It is believed that flow step focusing can operate on materials havingelectrophoretic mobility, to include particles, nanoparticles, and cellsas well as molecules dissolved in solution. Such dissolved or suspendedmaterials will either have a charge, or could be given one by complexingthem with buffer additives.

The position of the band of interest may be determined using varioustechniques, such as fluorescence. After the band becomes sufficientlyconcentrated, other techniques such as optical absorbance or electricalconductimetry could be used. In one embodiment, an automated systemreceives the position of the band and regulates the pressure and/or thevoltage, for example to direct the band towards or away from the sidechannel.

To find the location of a band, a detector could be employed on theoutput of the side channel: scanning through electric field strengthsand/or flow rates would sequentially proceed band positions past theside channel outlet to be detected. You'll see a spike as each onepasses. This embodiment could have applications such as in proteomics,wherein the output is analyzed by, e.g., a mass spectrometer (MS). In afurther embodiment, proteins in the output could be digested (such as bypassing through a packed bed of immobilized trypsin) so thatconcentrated bands of proteins are sequentially digested and analyzed byMS for sequencing, identification, and/or quantitation.

Examples

A proof-of-concept device was fabricated in polymethylmethacrylate(PMMA) using mechanical machining and laser ablation. It included a“cross” intersection of a side channel with a main channel. The mainchannel was 100 μm wide and 60 mm long. The side channels met the mainchannel at right angles in its middle. The use of two side channels isoptional, and in some embodiments one side channel could be used. In theexemplary device, two side channels were used in to provide robustnessagainst clogging. In some experiments, clogging of one side channel didoccur and the experiment continued with only one functional sidechannel. The side channels were 20 μm wide and 20 mm long. All channelswere 20 tm deep. All channels terminated in 6.35 mm diameter wells,accessible from the top. FIG. 1 is a schematic overhead view of anexemplary channel configuration for flow step focusing.

Well caps were fashioned from pipette tips in an arrangement depicted inFIG. 2. These allowed the airspace over the fluid in each well to besealed and connected to a pressure controller. The caps on the ends ofthe main channel were also equipped with a length of platinum wire thathung into the fluid to provide the electrical connections. Pressure wasprovided via silicone tubing from the outlets of the pressurecontrollers (Alicat Scientific, Tucson, Ariz.).

A positive voltage was applied to the inlet chamber and the outletchamber was grounded. The side chambers were allowed to floatelectrically. A positive pressure was applied to the inlet chamber. Theside chambers were connected to a single pressure controller andmaintained at a pressure above ambient, but below the inlet pressure. Inthis way the amount of fluid being removed at the intersection could becarefully controlled. Typical values were inlet pressure of 11 mbar,side channel pressure of 5.7 mbar, outlet at ambient pressure, and 500Vapplied voltage. No attempts were made to suppress electroosmosis.Instead, pressures were adjusted to counteract it and produce thedesired net flows.

Negatively charged solutes undergo electrophoretic migration toward theinlet, but are driven downstream by the bulk flow. Their net velocity isthe sum of two velocities: the mean flow velocity downstream and theelectrophoretic velocity upstream. At the intersection, a fraction ofthe bulk flow is lost to the side channels, so that the flow downstreamof the intersection is reduced. Under appropriate values of voltage andpressures, there will be a window of mobility in which a molecule ofinterest will have a net downstream velocity in the region upstream ofthe intersection and a net upstream velocity in the region downstream ofthe intersection. As a consequence, the ultimate disposition of that amolecule will be to travel out through the side channel(s).

When the molecule of interest has a mobility that falls within thewindow, it will become concentrated in the region of the intersectionand be pulled out through the side channels. The degree of concentrationis simply the ratio of the volumetric flow rates in the main and sidechannels, respectively, and thus can be controlled.

A different phenomenon occurs when it is one of the buffer componentsthat is selectively removed. Buffer components may be as much as sixorders of magnitude more concentrated than the molecule of interestand/or other analytes. When a buffer ion is selectively removed,significant charge separation is quickly established. If, for example,the borate anion is selectively removed from a Tris-Borate buffer, thereis a buildup of negative charge in the side channel and a buildup ofexcess positive charge from the unpaired Tris in the main channel. Theelectric fields created by the charge separation drive the ionselectrophoretically to counteract the selective removal, and anequilibrium state is soon established, where the secondary electricfields prevent the buffer molecule from being continuously removed. Thesecondary effect of this is to also perturb the electric field in themain channel so that they are no longer uniform. In the non-uniformelectric field, it becomes possible to focus solute molecules at anypoint in a large length of the channel. Because the solutes can befocused well away from the intersection, they are not continuouslyremoved as they enter the concentrated region, and the achievableconcentration is no longer limited by the relative flow rates in the twochannels. The position of the concentrated band can be moved around bychanging either the pressure applied to the inlet channel or the appliedfield. When the band passes the intersection, it is pulled into the sidechannel.

FIG. 3 is a series of images taken sequentially (top to bottom) of aband moving from right to left across an intersection with a sidechannel. The initially strong band is reduced as solute is removed atthe intersection.

In one embodiment, the system separates solutes based on electrophoreticmobility primarily or entirely. One could alter or enhance theselectivity by adding psuedostationary phases, such as cyclodextrins ormicelles. In this way, neutral analytes could be concentrated, andsolutes with similar electrophoretic mobility could be separated.

In order to remove the band without perturbing the focusing method, across intersection could be used. Solution can be removed from one sidechannel while an equal amount of replacement buffer is added from theopposite side channel. This prevents a change in the flow velocity assolution passes the intersection, but could be used to changeconductivity or other features of the buffering solution

The described technique provides several advantages, including:

Simplicity: The system requires only control of voltage and pressure,and requires no moving parts within the “chip” making up the channels.Inexpensive and reliable solenoid valves can be used within pressurecontrollers to regulate flow.

Adaptability: Unlike free-flow IEF, where solutes flow through thechannel at a constant velocity and are only retained briefly beforebeing flushed out, the described technique traps the solutes within aband in the channel until a time such as the band is moved across theintersection. As a result the concentration factor can be adapted byholding the band for varying amounts of time before samples arecollected. Significantly, the collection time can be adapted in realtime in response to changes in concentration of the sample stream, sothat the detection limit and range of a detection system including thedevice can be adapted to meet the needs of the sample.

Speed: Concentration and purification take place simultaneously, ratherthan sequentially in a multistep process

Cost: The voltage source and pressure supply can be made with currentoff-the-shelf components.

All documents mentioned herein are hereby incorporated by reference forthe purpose of disclosing and describing the particular materials andmethodologies for which the document was cited.

Although the present invention has been described in connection withpreferred embodiments thereof, it will be appreciated by those skilledin the art that additions, deletions, modifications, and substitutionsnot specifically described may be made without departing from the spiritand scope of the invention. Terminology used herein should not beconstrued as being “means-plus-function” language unless the term“means” is expressly used in association therewith.

REFERENCES

Each of the following referenced documents is incorporated by referenceherein in its entirety:

Electric Field Gradient Focusing

-   -   Huang, Z, and C F Ivory. 1999. Digitally controlled        electrophoretic focusing. Analytical Chemistry 71, no. 8:        1628-1632.    -   Petsev, Dimiter N, Gabriel P Lopez, Cornelius F Ivory, and Scott        S Sibbett. 2005. Microchannel protein separation by electric        field gradient focusing. Lab on a chip 5, no. 6 (June): 587-97.    -   Lin, S L, H D Tolley, and M L Lee. 2005. Voltage-controlled        electric field gradient focusing with online UV detection for        analysis of proteins. CHROMATOGRAPHIA 62, no. 5-6 (September):        277-281.

Temperature Gradient Focusing

-   -   Balss, K M, D Ross, H C Begley, K G Olsen, and M J Tarlov. 2004.        DNA hybridization assays using temperature gradient focusing and        peptide nucleic acids. Journal of the American Chemical Society        126, no. 41: 13474-13479.    -   Tang, G Y, and C Yang. 2008. Joule heating induced temperature        gradient focusing in a microfluidic channel with a sudden change        in cross section. Proceedings of the Micro/Nanoscale Heat        Transfer International Conference 2008, Pts a and B: 179-184.

Isoelectric Focusing

-   -   Huang, Z, and C F Ivory. 1999. Digitally controlled        electrophoretic focusing. Analytical Chemistry 71, no. 8:        1628-1632.    -   Ivory, C F. 2000. Brief Review of Alternative Electrofocusing        Techniques. Separation Science and Technology, 35(11):        1777-1793.    -   Wen, J, E W Wilker, M B Yaffe, and K F Jensen. 2010.        Microfluidic Preparative Free-Flow Isoelectric Focusing: System        Optimization for Protein Complex Separation. Analytical        Chemistry 82, no. 4: 1253-1260.

What is claimed is:
 1. An apparatus configured to isolate andconcentrate a molecule of interest, the apparatus comprising: a mainchannel having an inlet and an outlet; a side channel intersecting themain channel and having a side channel outlet; a first pressurecontroller operably connected to the inlet and configured for pumpingfluid into the inlet; a second pressure controller operably connected toeither the main channel outlet or the side channel outlet and configuredto apply a pressure thereto; and electrodes at each of the inlet and theoutlet operably connected to a voltage controller configured to generatea voltage between the electrodes so as to generate an electric fieldalong the channel, wherein the electric field is sufficiently strong todrive a molecule of interest in a direction opposite to flow of a liquidfrom the inlet to the outlet.
 2. An apparatus according to claim 1,wherein the main channel is substantially optically transparent at theintersection with the side channel.
 3. The apparatus according to claim1, further comprising a second side channel positioned in the vicinityof the side channel and configured to add fluid to the main channel. 4.The apparatus according to claim 1, further comprising a detectorconfigured to detect a position of a band of a molecule of interest inthe main channel.
 5. The apparatus according to claim 4, furthercomprising an automated system configured to receive the position of theband from the detector and to adjust the first pressure controllerand/or the voltage controller in response thereto.
 6. The apparatusaccording to claim 1, further comprising an analytic instrument operablyconnected to the side channel outlet.
 7. The apparatus according toclaim 6, wherein said analytic instrument is a mass spectrometer.
 8. Theapparatus according to claim 6, further comprising a protein digesterdownstream of the side channel outlet.
 9. An apparatus configured toisolate and concentrate a molecule of interest, the apparatuscomprising: a main channel having an inlet and an outlet; a side channelintersecting the main channel and having a side channel outlet; a firstpressure controller operably connected to the inlet and configured forpumping fluid into the inlet; a second pressure controller operablyconnected to either the main channel outlet or the side channel outletand configured to apply a pressure thereto; electrodes at each of theinlet and the outlet operably connected to a voltage controllerconfigured to generate a voltage between the electrodes so as togenerate an electric field along the channel, wherein the electric fieldis sufficiently strong to drive a molecule of interest in a directionopposite to flow of a liquid from the inlet to the outlet; and adetector configured to detect a position of a band of a molecule ofinterest in the main channel, the detector operating through a region ofthe main channel at the intersection with the side channel that issubstantially optically transparent.